Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts.
Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts.
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DOI：
10.1038/s41596-021-00516-2
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发表时间：
2021-05
期刊：
影响因子：
14.8
通讯作者：
Hainer SJ
中科院分区：
文献类型：
--
作者：
Patty BJ;Hainer SJ
确定染色质相关蛋白在整个基因组中的定位，为dna结合蛋白的功能及其与疾病的联系提供了见解。然而，需要大量细胞或组织样本的既定方案目前限制了该领域临床和生物医学研究的应用。此外，大多数技术已被优化用于评估丰富的组蛋白定位，禁止在低细胞数量下研究非组蛋白定位。我们最近描述了一种在低至一个细胞中分析染色质相关蛋白定位的方案：在靶标下进行超低输入切割并使用核酸酶（uliCUT&RUN）释放。uliCUT&RUN是一种基于拴链酶的方案，利用重组蛋白、抗体识别和严格的纯化相结合，选择性地靶向感兴趣的蛋白质并分离相关的DNA。在自然条件下进行，uliCUT&RUN以低输入和高精度描述蛋白质定位到染色质。与其他分析技术相比，uliCUT&RUN可以确定低细胞数量下的非组蛋白染色质占用率，从而允许在罕见样品中研究一系列dna结合蛋白的分子功能。从样品制备到测序文库提交，uliCUT&RUN方案执行时间小于2天，随附的数据分析时间表取决于经验水平。
Determining chromatin-associated protein localization across the genome has provided insight into the functions of DNA-binding proteins and their connections to disease. However, established protocols requiring large quantities of cell or tissue samples currently limit applications for clinical and biomedical research in this field. Furthermore, most technologies have been optimized to assess abundant histone protein localization, prohibiting the investigation of nonhistone protein localization in low cell numbers. We recently described a protocol to profile chromatin-associated protein localization in as low as one cell: ultra-low-input cleavage under targets and release using nuclease (uliCUT&RUN). Optimized from chromatin immunocleavage and CUT&RUN, uliCUT&RUN is a tethered enzyme-based protocol that utilizes a combination of recombinant protein, antibody recognition and stringent purification to selectively target proteins of interest and isolate the associated DNA. Performed in native conditions, uliCUT&RUN profiles protein localization to chromatin with low input and high precision. Compared with other profiling technologies, uliCUT&RUN can determine nonhistone protein chromatin occupancies in low cell numbers, permitting the investigation into the molecular functions of a range of DNA-binding proteins within rare samples. From sample preparation to sequencing library submission, the uliCUT&RUN protocol takes <2 d to perform, with the accompanying data analysis timeline dependent on experience level.
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剪切染色质长度对ChIP-seq质量和灵敏度的影响。
DOI：
10.1093/g3journal/jkab101
发表时间：
2021-06-17
期刊：
G3 (Bethesda, Md.)
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